Real-time imaging of cellular function in vivo and of cell/tissue localization can be achieved with high sensitivity and specificity by using fluorescent probes together with fluorescence and confocal microscopy. For example, following entry of the probe DCFH2-DA into the cell it is converted by intracellular esterases to DCFH2, which upon oxidation by free radicals, mainly •OH, CO3 •-, NO2 •, and thyl radicals (such as GS•), yields the fluorescent product (DCF) (reviewed by ). Nitrogen oxide is produced at low concentrations and has a short half-life, which makes it difficult to detect in vivo. Interest in NO, due to its ubiquity and physiological
relevance, has therefore led to the generation of several techniques for measuring its production. STA-9090 For example, the rapid reaction of 2,3-diaminonaphthalene (DAN) with NO to form the fluorescent product 1-(H)-naphthotriazole (NAT) is the basis for a very sensitive
analytical method to measure AZD1480 concentration NO production. DAN does not react directly with NO and therefore does not inhibit its actions. The high sensitivity of this technique allows its use in the quantification of NO production in living cells [23–25]. However, perhaps the most commonly employed methods for the analysis of NO in aqueous solutions is by measuring NO2 – using the Griess reagent . Alternatively, inhibitors of NO function can also be used to understand the physiological roles of this
molecule. Carboxy-PTIO (c-PTIO) is a water-soluble and stable free radical that reacts stoichiometrically with NO. In vivo, c-PTIO inhibits the physiological effects mediated by NO, whereas in vitro it can be used to quantitate NO levels by ESR spectrometry . The lichen Ramalina farinacea (L.) Ach. is a widespread species with large environmental tolerance. This green-greyish lichen is a fruticose, pendulous, epiphytic species Vasopressin Receptor that is very common in Mediterranean sclerophyllous oak forests. It lives on a great variety of substrates and different habitats such as plant bark, decomposing wood and rocks . In the Iberian Peninsula it occurs at all altitudes, more frequently in areas with regular fogs being absent in maritime habitats. It shows especial preference for places with a high atmospheric humidity. This lichen is the Ramalina species with lower sensitivity to SO2 and is considered as toxitolerant . The aim of this work is to investigate the release and role of NO in the oxidative stress caused by rehydration in the lichen Ramalina farinacea (L.) Ach. NO and ROS specific fluorescent probes will be used to morphologically localize these molecules in vivo with fluorescence and confocal microscopy. Furthermore, ROS kinetics and chlorophyll autofluorescence will be recorded during the first minutes after rehydration. Lipid peroxidation and NO-endproducts will be quantified at different time points.